African Journal of Biotechnology Vol. 5 (11), pp. 1161-1166, 2 June 2006 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2006 Academic Journals

Full Length Research Paper

Rearing the house fly predator pumilio (Erichson) (Coleoptera: ) on an artificial diet

K. A. Achiano and J. H. Giliomee*

Department of Botany and Zoology, University of Stellenbosch, Private Bag X1, Matieland, 7602 South Africa.

Accepted 23 March, 2006

A number of concentrations of PRO-PLEX™, a protein rich food additive, were incorporated into an artificial diet to determine its effect on the predatory , (Erichson). The impact of this host-free artificial diet on developmental time, larval weight, oviposition and mortality was assessed. When C. pumilio is fed on a host-free diet, the total development of egg to adult was prolonged in comparison with those reared on natural diet by an average of 3.95 ± 0.17 days. The number of stadia was not affected by the artificial diet. The increase in weight of both 1st and 2nd instar larvae was highly significantly and strongly correlated with the concentration of PRO-PLEX™ in the artificial diet (P < 0.001). Adults fed on the artificial diet laid eggs, but significantly less than on the natural diet (P < 0.05). C. pumilio larvae reared on a natural diet had a significantly faster rate of oviposition, shorter developmental time and lower mortality as compared to those fed on the artificial diet.

Key words: Artificial diet, natural diet, Carcinops pumilio, body weight, developmental time, mortality.

INTRODUCTION

Rearing on artificial diets has many advantages over periods when natural prey is scarce or unavailable over natural food, particularly in the case of parasitoids for rearing, a suggestion also made by Hattingh (1991). and predators where the need to provide prey is The first known example of mass rearing an on eliminated. This should reduce the cost of production, an artificial diet was the larvae of the blowfly, Cochliomyia one of the problems limiting the use of insect parasitoids hominivorax (Coquerel) in 1936. This was a hallmark, and predators in inundative releases (Leppla, 1996; which facilitated suppression of screwworm by genetic Thompson, 1999). Thus to overcome such problems and means (Knipling, 1984). Excellent artificial diets are now meet the demands for large numbers of insects required available for rearing many phytophygous , but for fundamental research in the fields of physiology, are generally lacking for entomophagous arthropods ecology, genetics and insect control techniques such as (House, 1977; Singh, 1977). The house fly is an male sterilization and integrated pest management (IPM) unsuitable prey source for mass production of its programs, the use of artificial diets for rearing insects has predators such as Carcinops pumilio (Erichson), because generated great interest since the 1950’s (Singh, 1977). larvae that escape predation disrupt the substrate and Mass reared insects on an artificial diet could also serve interfere with pupating C. pumilio larvae (Geden, 1990). as a cheap source of food for in zoos and birds, However, alternative prey can be used, such as such as poultry. Endangered insect species could be sphaerocerid flies (Geden, 1984), Drosophila repleta reared on an artificial diet in the laboratory and later (Wallaston) (Fletcher et al., 1991) and D. melanogaster released into their own natural habitat (Singh, 1977). The (Meigen) (Achiano and Giliomee, unpublished). Geden artificial diet could also be used as a dietary supplement (1990) stated that cost-effective C. pumilio production must await the development of artificial diets and methods of handling that will minimize the effects of cannibalism in culture. *Corresponding authors E-mail: [email protected]. Tel: 272180827 The house fly, Musca domestica L. is a serious 18. Fax: 27218082405. cosmopolitan pest. Poultry farmers employ various

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techniques to control them but rely heavily on the use of The open portion of the container was later covered with organdy chemicals which constitutes a significant production held in place with a rubber band. It was then placed in a cool room expense (Lazarus et al., 1989). Such heavy reliance on at 5 ± 1°C until use. On the day of use, several pieces of about 10 g of diet were transferred into each rearing container. The shelf life of insecticides for pest suppression has the serious the diet was not determined but used diet stored for 30 days in drawback of the target pest becoming resistant (Rutz and similar experiments gave similar results to those of fresh diet. Scott, 1990; Howard and Wall, 1996) and the possible destruction of beneficial insects (Theiling and Croft, 1988). This could create new secondary pests and a later Preliminary experiments resurgence of damaging population levels of the primary In order to reduce the disturbance of the different stages during the pest (Metcalf and Luckmann, 1975). One way of final observations on development, preliminary development times overcoming such problems is to develop an IPM program were determined for the stages from egg to hatch, 1st instar larva to with emphasis on biological control. In such a programme 2nd instar larva and 2nd instar larva to adult emergence from the the availability of an artificial medium for rearing the pupa, at each protein concentration level from 30 to 70%. To obtain eggs, freshly emerged adult were placed on the artificial principal predator, C. pumilio, would be of great diet for 10 days at each of the different concentration levels. advantage. However, an economical mass rearing Several pieces of the artificial diet at each protein concentration method on an artificial diet is yet to be developed for C. level were mixed with 1 litre of moistened bran. The bran to water pumilio. ratio was 1: 2.9 (w/w) which produced about 60-65% moisture In an attempt to find an artificial diet for C. pumilio, content. The mixture was then transferred into 2 litre milk PRO-PLEX™, a protein rich food additive used by body containers. The newly emerged adult beetles that were kept on the artificial diet were placed on the bran and protein mixture for 24 h. builders, was supplied. When supplied with PRO-PLEX™ They were then removed to prevent them from eating their eggs. powder, C. pumilio attempted to consume it but could not Five replicates were used. The 2 litre container with the top hold on with the mandibles. The powder adhered to the removed was covered with organdy held in place with a rubber body or it got entangled in the melted powder. It was band. The preliminary tests were observed daily to obtain estimates therefore decided to investigate the possibility of of the above developmental periods for each concentration level. formulating PRO-PLEX™ powder in another form that will Similar procedures were also followed for C. pumilio fed on Drosophila melanogaster as a source of natural diet. enhance the ability of C. pumilio to feed on it. The objectives of this study were therefore: (1) to formulate an artificial diet devoid of any prey material using PRO- Developmental times of stages PLEX™ and (2) to evaluate the nutritional and physical The developmental times of egg to 1st instar larva, 1st instar to 2nd adequacy of the diet formulation using (a) developmental nd instar larva and 2 instar larva to adult emergence from the pupa time, (b) mortality rate and (c) ovipositon rate as were determined. The media and the procedure of rearing parameters. described above were used in 5 replicates for each protein concentration. After 24 h, the adults were removed and the media of each replicate was divided into 20 test tubes to facilitate easier MATERIALS AND METHODS counting of the larvae. The test tubes, covered with a plastic lid with small holes pierced into it using an office pin, were placed in an Insects incubator at 30 ± 1°C. From two days before the estimated time of egg hatch, based on the preliminary experiment described above, the media were examined for the presence of 1st instar larvae. The adults of C. pumilio used in this study were from a population st maintained in the laboratory, originating from samples obtained The 1 instar larvae removed from each test tube were introduced into new medium with the appropriate protein from Stellenbosch University Experimental Poultry Farm, nd Elsenburg, Western Cape Province, South Africa (33° 51´S; 18° concentration. Two days before the appearance of the 2 instar 50´E). All experiments were carried out in an incubator set at 30 ± larvae was expected, the test tubes were examined at 24 h 1°C at a Stellenbosch University Laboratory (33° 54´S; 18° 57´E). intervals to detect their presence. The developmental time of the 2nd instar larvae to adults was Beetles used in the experiment were F6 progeny. st determined by observing the same individuals used in the 1 instar larvae studies. All test tubes were observed for adults as described above, except that a rotational method was used to reduce the Diet preparation disturbance of the pupae. The data for developmental time (days) of various stages was The dietary composition of the PRO-PLEX™, designated as analyzed using ANOVA. The means were separated at p < 0.05 “protein” in this study is listed in Table 1. Diets were prepared using level by Fisher’s LSD. protein and agar in various ratios (w/w) so that diets with 30, 40, 50, 60, and 70% protein were obtained. Each mixture was blended for 60 seconds and brought to boil for 5 to 10 min whilst continuously Weight measurement stirring. After it had boiled for 5 minu, 1.5 g Na-methylhydroxy benzoate dissolved in 2 ml alcohol was added to each mixture to The wet weight of ten 1st and 2nd instar larvae from each protein prevent fungal growth in the medium. After preparation, while the concentration level of individuals obtained was measured using a diet which had a rather smooth texture was still hot, it was poured Sartorius microbalance which weighed to the nearest 0.01 mg. The into 1 litre plastic containers. The containers were left open for 10 larvae were weighed at the beginning of each stage. The effects of min to allow some moisture to evaporate, to cool down and solidify. st nd ® diet concentration on weight of 1 and 2 instar larvae were The top of the solidified medium was then covered with Parafilm . analyzed by regression.

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Table 1. Components of PRO-PLEX™ in mg/100 g. Mortality rates

To determine the mortality of 1st instar larvae, five newly hatched Nutritional information* unit larvae were transferred onto the medium in test tubes using a fine Vitamin A, B & C 0.06 wet camel’s hair brush. There were 20 replicates for each protein nd Nicotinamide 0.71 concentration of 30, 40, 50, 60 and 70%. Individual 2 instar larvae obtained from surviving 1st instar larvae were used to determine the Calcium pantothenate 1.90 mortality rate from 2nd instar to adult emergence. Five 2nd instar Phosphorus 171.00 larvae per test tube and ten replicates for each concentration level st nd Calcium 78.00 were used. The percentage mortality from 1 to 2 instar larva and 2nd instar larva to adult emergence was calculated as [ x number Magnesium 84.00 dead / number of larvae per container] x 100. Copper sulphate 0.16 Sodium 1279.00 Oviposition Pottasium 1183.00 Protein (Albumin) 83000.00 The adults reared on the artificial diet at different concentrations Aspartame 400.00 described above were used to determine ovipositon rate. Two pairs of adult males and females from each protein concentration level of Essential amino acids unit 30, 40, 50, 60 and 70% were selected immediately after emergence L - Histidine 2403 and were placed in a small plastic container of 5 cm in height and 3 L - Isoleucine 2929 cm in diameter. The lid had a hole of 1.5 cm in diameter and this was covered with organdy, held in place with glue. This was L - Leucine 6833 replicated five times for each concentration. The adults were L - Lysine 5869 supplied with artificial diet ad libitum, with a small amount of moistened bran to serve as an oviposition site. After 24 h, they L - Methionine 3672 were removed and placed in a new container with fresh medium L - Phenylalanine 5009 and source of food. This routine was followed for ten days. Six days L - Threonine 3927 after the first introduction of the adults the media were examined for the presence of larvae and those found were counted. In this study, L - Tryptophan 1196 it was assumed that the number of larvae hatched was equal to the L - Valine 4142 eggs oviposited and that no mortality occurred in the egg stage. The data for oviposition rates was analyzed using ANOVA. The means were separated at P < 0.05 level by Fisher’s LSD.

0.10 RESULTS 3

- a 0 1

/ ) 0.08 g m Developmental time (

e y = 0.001x - 0.006 a 0.06 2 v r R = 0.988 a l r P < 0.001 Developmental times of the various stages differed a t 0.04 s n i x significantly between the artificial diet and natural diet (P t s 1

< 0.05) (Table 2). C. pumilio reared on the artificial diet f 0.02 o t h developed more slowly than those reared on natural diet. g i st nd e 0.00 The developmental time from 1 to 2 instar larva in W 30 40 50 60 70 relation to the protein concentration or natural diet % protein in artificial diet followed no particular pattern. The developmental time nd b y = 0.004x - 0.040 from the 2 instar larvae to adult stage was also R2 = 0.881 significantly shorter on the natural diet as compared to

3 P < 0.001 - larvae fed on artificial diet. The total developmental time 0 1 / )

g 0.40 from egg to adult of C. pumilio fed on artificial diet was m ( longer than that for C. pumilio fed on natural diet (Table e

a 0.30 v r 2). The number of stadia was not affected by the artificial a l

r x

a 0.20 t diet. s n i

d 0.10 n

2

f o

t 0.00 Effect of artificial diet on larval weight g h i

e 30 40 50 60 70

W st % protein in artificial diet The 1 instar larvae that emerged from eggs of adult C.

pumilio fed on the artificial diet at the higher protein Figure 1. Mean weight of a = 1st instars; b = 2nd instars reared on concentrations were heavier than those fed on natural five different protein concentration levels of artificial diet. X = diet (Figure 1). However, the 1st instar larvae of adults fed weight of larvae fed on natural diet. on the natural and artificial diet with a protein

1164 Afr. J. Biotechnol.

Table 2. Developmental time of various stages of C. pumilio fed on natural and artificial diets.

% protein in Developmental time (days) (mean ± SE.)* Total artificial diet Egg - 1st instars larva 1st instars - 2nd instar larva 2nd instar - adult developmental time 30 5.2 ± 0.09 b 4.70 ± 0.16 a 13.50 ± 0.17 b 23. 40 ± 0.16 b 40 5.0 ± 0.01 b 5.30 ± 0.11 b 13.85 ± 0.31 b 24.15 ± 0.21 b 50 5.1 ± 0.07 b 4.55 ± 0.11 a 15.95 ± 0.44 c 25.60 ± 0.27 c 60 5.0 ± 0.06 b 5.40 ± 0.11 b 16.60 ± 0.78 c 27.00 ± 0.34 c 70 5.0 ± 0.09 b 4.40 ± 0.11 a 15.70 ± 0.50 c 25.10 ± 0.30 c Natural diet 3.8 ± 0.02 a 5.1 ± 0.10 b 11.3 ± 0.15 a 20.10 ± 0.11 a

*Figures followed by the same letters are not significantly different; P < 0.05

Table 3. Mortality rates of C. pumilio on an artificial and natural diet.

% mortality % protein 1st to 2nd instar larvae* 2nd instar larvae to adult emergence** 30 95.5 a 96.0 a 40 92.0 a 94.0 a 50 92.5 a 76.0 a 60 89.0 a 66.0 a 70 86.0 a 70.0 a Natural diet 22.0 b 16.0 b

N = 10, 5 larvae/container* N = 10, 5 larvae/container** Figures with the same letters are not significantly different. P < 0.05

Table 4. Oviposition rates (eggs/female/day) of C. pumilio on an Effect of artificial diet on mortality artificial and natural diet. The mortality of 1st and 2nd instar larvae reared on the Oviposition Oviposition artificial diet was extremely high, exceeding 85% and -1 % artificial diet* (mean ± SE.)** day /female 65%, respectively, whilst the mortality of larvae reared on 30 0.9 ± 0.41 a 0.09 natural diet was comparatively low at 22% and 16%, respectively. The mortality of both 1st and 2nd instar larvae 40 2.7 ± 0.50 a 0.27 reared on artificial diet was four times more than those 50 1.5 ± 0.50 a 0.15 reared on natural diet (Table 3).

60 2.0 ± 0.49 a 0.20 Effect of artificial diet on oviposition 70 1.0 ± 0.30 a 0.10 From the oviposition rate as a measure of the Natural diet 7.5 ± 0.70 b 1.50 reproductive potential of C. pumilio reared on the artificial *N = 10. **Figures with the same letters are not significantly diet, it was clear that adults fed on the natural diet had different; P < 0.05. the highest oviposition rate of 7.5 eggs per day. This differed significantly from the oviposition rate of adults fed on artificial diet at all concentration levels (P < 0.05)

nd (Table 4). There was no significant difference in the rate concentration of 30% were the same weight. The 2 in of oviposition between the various protein concentrations. star larvae fed on 70% protein were also heavier than those fed on the natural diet. At the lower protein DISCUSSION concentrations the differences were not significant except at the lowest concentration (30%) where the larvae were The developmental time for an insect reared on an significantly lighter. The increase in weight of both 1st and artificial diet is often prolonged, as was the case with C. 2nd instar larvae were highly significantly and strongly pumilio. This was also found by De Clercq and Degheele correlated with the protein concentration in the artificial (1992) and Wittmeyer and Coudron (2001) for Podisus diet (P < 0.001) (Figure 1). maculiventris (Say), a generalist stinkbug predator, and

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Carpenter and Greany (1998) and Gelman et al. (2000) step towards finding an artificial diet that would allow for Diapetimorpha introita (Cresson), a parasitoid wasp. continuous rearing of C. pumilio, ensuring its availability The prolongation of the developmental time of C. pumilio at all times for utilization in the biological control of house was most pronounced during the last stadium. This flies. information provides direction for future research on improving this artificial diet to reduce developmental time. Furthermore, there were a small number of individuals ACKNOWLEDGEMENTS that developed faster than others, indicating they adapted more quickly to the diet. From such fast developers a The authors wish to thank Dr K. L. Pringle of the strain could be selected that are more suited to the Department of Entomology and Nematology, University of artificial diet formulated, a suggestion also made by Stellenbosch, South Africa for his comments and inputs. Wittmeyer and Coudron (2001) for P. muculiventris. Financial assistance was received from the University of At certain protein concentrations, the weight of the 1st Stellenbosch and the National Research Foundation. and 2nd instar larvae was similar or higher than those fed on natural diet, indicating that they developed adequately on the artificial diet. However, adult females that REFERENCES developed from larvae reared on the artificial diet had a Brewer FD, Lindig O (1984). Ingredients for insect diets. In: King, EG, significantly lower rate of oviposition than those reared on Leppla N C Advances and challenges in insect rearing. USDA, Agric. the natural diet. From the weight alone, one would have Res. Service, New Orleans. expected the females from the artificial diet to produce Carpenter JE, Greany PD (1998). 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